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Over the past few years we have developed the ability to acquire images through a confocal microscope that contain, for each pixel, the simultaneous fluorescence lifetime and spectra of multiple fluorophores within that pixel. We have demonstrated that our system has the sensitivity to make these measurements on single molecules. The spectra and lifetimes of fluorophores bound to complex molecules contain a wealth of information on the conformational dynamics and local chemical environments of the molecules. However, the detailed record of spectral and temporal information our system provides from fluorophores in single molecules has not been previously available. Therefore, we ...
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Description

Over the past few years we have developed the ability to acquire images through a confocal microscope that contain, for each pixel, the simultaneous fluorescence lifetime and spectra of multiple fluorophores within that pixel. We have demonstrated that our system has the sensitivity to make these measurements on single molecules. The spectra and lifetimes of fluorophores bound to complex molecules contain a wealth of information on the conformational dynamics and local chemical environments of the molecules. However, the detailed record of spectral and temporal information our system provides from fluorophores in single molecules has not been previously available. Therefore, we have studied several fluorophores and simple fluorophore-molecule systems that are representative of the use of fluorophores in biological systems. Experiments include studies of a simple fluorescence resonance energy transfer (FRET) system, green fluorescent probe variants and quantum dots. This work is intended to provide a basis for understanding how fluorophores report on the chemistry of more complex biological molecules.

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